In single molecule Frster resonance energy transfer (FRET) spectroscopy, kinetics and dynamics of molecular processes are typically determined by analyzing energy transfer efficiency calculated using fluorescence intensity from donor and acceptor dyes. To understand complex molecular interactions and conformational changes during binding and oligomerization processes, it is important to utilize all available information from single molecule measurements. Since FRET efficiency is related to the lifetimes of dyes, additional information of the process can be extracted by analyzing fluorescence intensity and lifetime together. From the correlation between the FRET efficiency and fluorescence lifetime in 2D FRET efficiency-lifetime distribution, it is possible to estimate conformational flexibility of protein molecules. We have developed single molecule fluorescence method that combines two- and three-color FRET and fluorescence lifetime analyses. We have applied this method to characterization of conformations of oligomers of the tetramerization domain (TD) of the tumor suppressor protein p53. TD is known to exist as a monomer at a low concentration and form a dimer and a tetramer sequentially as the concentration is raised. Our focus in this project is the characterization of the monomer and dimer for which atomic-resolution structures are not available. We have found that the tetramer dissociation constant is so low that individual oligomers cannot be clearly separated. Using single-molecule spectroscopy, it is possible to detect individual oligomers in equilibrium. Combination of two- and three-color FRET experiments and the fluorescence lifetime analysis allows for a quantitative comparison of the oligomer conformations. The monomer is intrinsically disordered as expected and the dimer conformation is very similar to that of the tetramer but the C-terminus of the dimer is less structured and more flexible. This development of single molecule fluorescence method will also be useful to study oligomerization of proteins that eventually for amyloid fibrils. As a first step in this project, we investigated monomer conformations of amyloid beta protein that is associated with Alzheimers disease. The monomer conformation of this protein has been studied using various methods including nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations. The consensus of conclusions of these studies is that the monomer is disordered. However, there is a controversy over the existence of residual structures that may be templates of the fibril formation. To address this controversy, we studied the two most abundant isoforms, the 40-residue (Abeta40) and 42-residue (Abeta42) peptides. Single molecule FRET experiments have a great advantage in studying Abeta monomers because the experiment is carried out at a very low protein concentration without complications from oligomer formation or aggregation. We observed that both isoforms behave like intrinsically disordered proteins with a characteristic conformational fluctuation time of 40 ns. In collaboration with Dr. Robert B. Bests group, we have complemented the FRET experiments with MD simulations using newly-developed force fields that properly characterize the dimension and dynamics of intrinsically disordered proteins. Both the experiments and simulations clearly show that both Abeta isoforms are largely disordered and there is an almost complete absence of states with stable secondary structures.